B cell receptor (BCR) triggered B cell activation is regulated by a variety of factors that control the outcome of the B cell's encounter with antigen including the developmental state of the B cell, the engagement of coreceptors and Toll-like receptors (TLR) and viral infection. Determining how these factors influence BCR-induced signaling should add fundamentally to our understanding of the mechanism by which B cells are activated. Over the last year we have made progress in defining the mechanisms by which the positive coreceptor, the CD19/CD21 complex, the inhibitory receptor, FcgammaRIIB1, and the TLR, TLR9 function to regulate signaling. [unreadable] [unreadable] The B cell coreceptors CD19/CD21 when coligated to the BCR through the binding of complement tagged antigens prolongs and enhances BCR signaling in part by prolonging the association of the BCR with sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts. We determined that the ability of the CD19/CD21 complex to function in rafts was dependent on a tetraspanin CD81 that is a component of the CD19/CD21 complex. Thus, in B cells from CD81-deficient mice and B cells expressing chimeric CD19 receptors that fail to associate with CD81, the CD19/CD21 complex when coligated to the BCR failed to stabilize the BCR in rafts. Many proteins that associate with lipid rafts do so by virtue of their acylation in particular by their palmitoylation, a reversible acylation event. We determined that CD81 becomes palmitoylated in the lipid rafts following crosslinking of the BCR and the CD19/CD21 complex and that palmitoylation is essential for the function of CD81. CD81 contains six cysteines within its cytoplasmic face that are potential sites of palmitoylation. To assess the contribution of these cysteines to CD81 function CD81 molecules containing mutated cysteine residues were expressed in B cell lines in which the endogenous wild type CD81 expression was knocked down by RNAi technology. These mutants showed defects in signaling following coligation of the BCR and the CD19/CD21 complex verifying the importance of these cysteine residues in the function of CD81. Constructs of CD81 that contained FRET donor fluorescent proteins were used in conjunction with FRET acceptor fluorescent proteins targeted to raft microdomains to demonstrate by FRET confocal microscopy in living cells that the cysteine residues of CD81 were essential for the association of CD81 with raft lipids following the coligation of the BCR and the CD19/CD21 complex. [unreadable] [unreadable] Progress was also made in determining how the FcgammaRIIB, a potent negative regulator of BCR signaling when coligated to the BCR, signals for apoptosis when crosslinked to itself. We learned that the FcgammaRIIB when crosslinked to itself becomes associated with lipid rafts and signals for apoptosis by a mechanism dependent on c-Abl but independent of both the phosphatase SHIP and the FcgammaRIIB?s ITIM motifs that are required for FcgammaRIIB?s inhibition of BCR signaling. Results of recent studies have provided evidence that crosslinking the FcgammaRIIB1 blocks the differentiation of memory B cells to antibody secreting plasma cells. Thus, the addition of mAb specific for FcgammaRIIB1 to human CD27+ memory B cells blocks the TLR-9 agonist-induced differentiation of memory B cells to plasma cells. These findings are significant in defining a new target for FcgammaRIIB1 mediated regulation. Studies are in progress to determine at which point in the differentiation of memory cells to plasma cells the FcgammaRIIB1 functions.[unreadable] [unreadable] Lastly, BCR signaling is greatly amplified by TLR-9 agonists. Recent studies have suggested that antigens that contain the TLR-9 agonist, CpG oligonucleotides, reduce the threshold for B cell activation and may, as a consequence, play a role in autoimmune disease by allowing the activation of low affinity self-reactive B cells. However, TLR-9 is only present in B cells in intracellular endosomal compartments and the cellular mechanisms by which CpG-containing antigens augment BCR signaling through TLR-9 are not well understood. Our recent studies using a combination of confocal and electron microscopy provided evidence that BCR signaling results in the recruitment of TLR9-containing endosomes to the intracellular multivesicular bodies to which the BCR transports CpG-containing antigens. These vesicles appear to be the site of augmented BCR/TLR-9 signaling. This novel mechanism for TLR-9 enhancement of BCR signaling may be important in providing new targets for therapy for autoimmune antibody responses.